The role of the Pi binding site in the mechanism of action of mitochondrial ATPase will be explored. Approaches will be made to identifying the subunit of the enzyme to which Pi binds by resolving the subunits of the beef heart enzyme as well as the subunits of an enzyme from a thermophilic bacterium. The simultaneous interaction of the enzyme with Pi, metals and oxyanions will be examined in a study of the binding of these three ligands by the beef enzyme. The nature of the interaction of Pi with mitochondrial ATPase in a higher state of aggregation, the oligomycin sensitive ATPase, will be studied by examining the binding of Pi to a highly purified preparation of this form of the enzyme.